Project Summary Title: Profiling chromatin-associated lncRNAs in numerous biological contexts. The human genome encodes thousands of long noncoding RNAs (lncRNAs), some of which are known to regulate important phenomena in the nucleus, such as dosage compensation, imprinting, and genome stability. It is currently unclear how many lncRNAs act on chromatin, but certainly many other examples of this paradigm remain to be discovered. With this motivation, I propose to develop, validate, and apply a biochemical method for rapidly identifying chromatin-associated lncRNAs, called Profiling of Interacting RNAs on Chromatin and sequencing (PIRCh-seq). To demonstrate proof-of-principle of this method, I have used PIRCh-seq to positively identify two lncRNAs in Drosophila (roX1 and roX2) that are known to interact with dosage compensated chromatin (H4K16ac-marked). I propose to further test PIRCh-seq by profiling lncRNAs that are associated with specific chromatin states (e.g. general chromatin, enhancers, heterochromatin) in Drosophila, and then validate these lncRNA candidates using complementary techniques, such as chromatin isolation by RNA purification and sequencing (ChIRP-seq). Next, I aim to apply PIRCh-seq to human cell lines, thus defining the sets of developmental-stage or cancer-specific chromatin-associated lncRNAs. Finally, from these resulting lncRNA catalogs, I will select several for further functional characterization towards understanding their mechanism of interaction with chromatin and possible roles in chromatin-templated process. For these experiments, I will use complementary genetic and biochemical techniques, including ChIRP-seq, CRISPR- enabled screening and genetic perturbation, and RNA structure probing. This research proposal will make deep impacts on the fields of lncRNA and chromatin biology: first, it will provide a powerful tool for discovering functional lncRNAs; second, it will identify lncRNAs associated with specific chromatin states in an important model organism and human cell types that are relevant to development and disease; lastly, it will discover novel examples of lncRNA?chromatin interaction that will deepen our understanding of lncRNA function.